mitogen-activated protein kinase 1 activates apoptosis during testicular ischemia–reperfusion injury in a nuclear factor-κB-independent manner

European Journal of Pharmacology 604 (2009) 27–35

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

Mitogen-activated protein kinase 3/mitogen-activated protein kinase 1 activates apoptosis during testicular ischemia–reperfusion injury in a nuclear factor-κB-independent manner Letteria Minutoli a, Pietro Antonuccio b, Francesca Polito a, Alessandra Bitto a, Francesco Squadrito a,⁎, Vincenzo Di Stefano a, Piero Antonio Nicotina c, Carmine Fazzari c, Daniele Maisano c, Carmelo Romeo b, Domenica Altavilla a a b c

Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, University of Messina, Italy Department of Medical and Surgical Pediatric Sciences, University of Messina, Italy Department of Human Pathology, University of Messina, Italy

a r t i c l e

i n f o

Article history: Received 24 July 2008 Received in revised form 24 November 2008 Accepted 9 December 2008 Available online 24 December 2008 Keywords: MAPK3/MAPK1 BAX Caspase 3 and 9 NF-κB Testis torsion

a b s t r a c t Nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase3/MAPK1 and MAPK8 are involved in testicular ischemia reperfusion injury (testicular-I/R). NF-κB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 activation and enhanced MAPK3/MAPK1 activity. To better understand the role of MAPK3/MAPK1 up-regulation during testicular-I/R, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO mice during testicular-I/R. KO and wild-type (WT) animals underwent 1 h testicular ischemia followed by 24 h reperfusion or a sham testicular-I/R. Animals received either PD98059 (5 mg/kg/ip) or its vehicle. MAPK3/MAPK1, BAX, caspase-3 and -9 and TNF-α expression were assessed along with histological examination and an immunostaining for protein of apoptosis. Testicular-I/R caused a greater increase in MAPK3/MAPK1 in KO than in WT animals in both testes. KO mice had a lower expression of the apoptotic proteins and TNF-α as well as reduced histological damage compared to WT. Immunostaining confirmed the lower expression of BAX in the Leydig cells of KO mice. Administration of PD98059, abrogated MAPK3/MAPK1 expression and slightly reduced TNF-α but did not improve or reverse the histological damage in KO. PD98059 significantly reduced the histological damage in WT mice and markedly reduced the apoptotic proteins in KO and WT mice. These results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-α, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-κB independent manner. These findings should contribute to better understand testicular torsion-induced damage. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Testicular torsion is a pathological condition which affects one out of 4000 males younger than 25 years. In humans, testicular torsion is a medical emergency requiring surgical intervention to counter-rotate the testis and spermatic cord to restore blood flow (Ringdahl and Teague, 2006). Restoration of proper blood supplies after repair testicular torsion, however, can also cause testicular ischemia–reperfusion

⁎ Corresponding author. Section of Pharmacology, Department of Experimental, and Clinical Medicine and Pharmacology, Torre Biologica, 5th floor, AOU Policlinico “G. Martino”, Via C. Valeria Gazzi, 98125 Messina, Italy. Tel.: +39 090 2213648; fax: +39 090 2213300. E-mail address: [email protected] (F. Squadrito). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.12.028

(testicular-I/R) injury which may impair testicular function and, as a consequence, impact fertility (Ringdahl and Teague, 2006). The mechanism underlying testicular damage after torsion has yet to be fully elucidate. In a mouse model of testicular-I/R we have previously shown that the mitogen-activated protein kinase (MAPK) family has a role in the pathogenesis of testicular ischemia–reperfusion injury (Minutoli et al., 2005; Antonuccio et al., 2006). MAPKs have been shown to induce the phosphorylation of several transcription factors activating up-stream regulatory elements of inducible genes involved in inflammation and apoptosis (Gaestel et al., 2007; Pimienta and Pascual, 2007). These findings suggest that a block of MAPKs activation might represent a potential therapeutic approach in the treatment of testicular torsion. Evolutionary conserved nuclear factor-kappaB (NF-κB) is a transcription factor which can be induced in response to environmental changes (Neumann and Naumann, 2007). NF-κB regulates a plethora

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of cellular pathways and processes including the immune response, inflammation, proliferation, apoptosis and calcium homeostasis (Egan and Toruner, 2006). As such, it represents a potential target for novel therapeutic strategies for a wide variety of diseases. Indeed, NF-κB has also been proposed to play a role in testicular ischemia (Lysiak et al., 2005) where it has been shown to increase expression of genes whose products mediate inflammatory and immune responses, including Tumour Necrosis Factor (TNF-α). We have shown previously that NF-κB and MAPK pathways interact to produce damage after testicular-I/R (Minutoli et al., 2007). NF-κB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 (also named JNK) and MAPK14 (also named p-38) activation and enhanced MAPK3/MAPK1 (also named ERK1/2) activity when compared to wild type animals. Since MAPKs are involved the activation of NF-κB (Barnes, 1997), it can be hypothesized that both MAPK8 and MAPK14 are severely blunted in mice lacking the NF-κB gene and may not be involve in KO mice following testicular ischemia–reperfusion. By contrast KO animals subjected to testicular-I/R showed a greater MAPK3/MAPK1 expression than in wild type mice suggesting several hypothetical explanation(s) for this finding: i) MAPK3/MAPK1 might stimulate additional molecular inflammatory pathways other than NF-κB; ii) higher expression of MAPK3/MAPK1 in KO mice might unmask a protective role for this kinase, as already shown for cardioprotection in the so-called reperfusion injury kinase (RISK) pathways (Hausenloy and Yellon, 2004); or iii) augmented MAPK3/ MAPK1 expression may serve as an up-stream signal to turn-on a programmed cell death in the testis, as already shown in heart (Dhingra et al., 2007). Activation of the apoptotic machinery has been shown in testicular torsion (Sun et al., 2008), with MAPK3/MAPK1, specifically, triggering apoptosis in several testicular cell lines (Sun et al., 2008). In the light of these findings, the aim of the present paper was to study the effects of PD98059, an inhibitor of MAPK3/MAPK1, on the expression of apoptotic proteins BAX, caspase 3 and 9 and TNF-α in KO mice during testicular-I/R with the goal of generating a more appropriate approach to the treatment of acute testicular torsion. Positive findings regarding the regulatory pathways and the effect of PD98059 on MAPK3/MAPK1 activation are presented in this experimental model. 2. Materials and methods 2.1. Animals and treatment All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the care and use of Laboratory animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Maryland). Breeding pairs of NF-kappaB KO mice (p105; b6; 129-Nfkb1fm1Bal; KO) and normal control littermates WT mice (C57Bl/6; 129; WT), both groups weighing between 22 and 30 g, were purchased from the Jackson Laboratory (Bar Harbor, Maine). The animals were provided a standard diet ad libitum and had free access to tap water. Mice were maintained on a 12-hour light/dark cycle at 21 °C. Animals were anesthetized with an intraperitoneal injection of 80 mg\kg of pentobarbital sodium to perform torsion of the left testis and spermatic cord, 720° for 1 h. The same testis was then detorted. Sham operated mice underwent the same surgical procedures as testicular-I/R mice except for testicular occlusion. We have used 84 rats randomly divided into four groups. Each group was composed of seven animals. Mice were randomized to receive either PD98059 (5 mg/kg/i.p) or its vehicle (1 ml/kg/i.p) immediately after detorsion. Sham animals received the same treatments with out torsion of the testis after surgery. Animals were sacrificed at the following time points: 30 min, 3 h and 24 h according to previous findings which showed peak expression of MAPKs at 30 min, TNF-α is at 3 h and peak histological damage at time 24 h (Minutoli et al., 2005). These same time points have also been reported for activation of apoptotic machinery (Sun et al., 2008).

2.2. Isolation of cytoplasmatic protein Briefly, pulverized testis sample were homogenized in 1 ml lysis buffer (25 mM Tris/HCl, pH 7.4, 1.0 mM EGTA,1.0 mM EDTA, 0.5 mM phenyl methylsulfonyl fluoride, aprotinin, leupeptin, pepstatin A (10 μg/ml each) and Na3VO4 100 mM, with a Dounce homogenizer. The homogenate was subjected to centrifugation at 15,000 g for 15 min. The supernatant was collected and used for protein determination using the bio-rad protein assay kit (Bio-Rad, Richmond, CA, USA). 2.3. Determination of MAPK3/MAPK1, TNF-α, BAX, caspase 3 and 9 by western blot analysis Protein sample (50 µg) were denatured in reducing buffer (62 mM Tris/HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.003% bromophenol blue) and separated by electrophoresis on an SDS (12%) polyacrylamide gel. The separated proteins were transferred on to a nitrocellulose membrane using the transfer buffer (39 mM glycine, 48 mM Tris/HCl, pH 8.3, 20% methanol) at 200 mA for 1 h. The membranes stained with Ponceau S (0.005% in 1% acetic acid) to confirm equal amounts of protein and were blocked with 5% non fat dry milk in TBS-0.1% Tween for 1 h at room temperature, washed three times for 10 min each in TBS-0.1% Tween, and incubated with a primary antibody for TNF-α, BAX, (Chemicon, Temecula, California) and for caspase 3 and 9 (Abcam, Cambridge, UK) and for the phosphorylated and total form of MAPK3/MAPK1 (Cell Signaling, Beverly, Massachusetts) in TBS-0.1% Tween overnight at 4 °C, diluted 1:1000. After being washed three times for 10 min each in TBS-0.1% Tween, the membranes were incubated with a secondary antibody peroxidase-conjugated goat anti-rabbit immunoglobulin G (Pierce, Rockford, Illinois) for 1 h at room temperature diluted 1:20000. After washing, the membranes were analyzed by the enhanced chemiluminescence's system according to the manufacturer's protocol (Amersham, Little Chalfont, UK). The MAPK3/MAPK1, TNF-α, BAX, caspase 3 and 9 protein signal was quantified by scanning densitometry using a bio-image analysis system (Bio-Profil Celbio, Milan, Italy). Equal loading of protein was assessed on stripped blots by immunodetection of β-actin with a rabbit monoclonal antibody (Cell Signaling, Beverly, Massachusetts) diluted 1:500 and peroxidaseconjugated goat anti-rabbit immunoglobulin G (Pierce, Rockford, Illinois) diluted 1:15000. All antibodies are purified by protein A and peptide affinity chromatography. 2.4. Histology Excised mouse testes were longitudinally sectioned and formalin fixed and paraffin embedded. Serial sections (5 µm) were stained with haematoxylin and eosin. Light microscopy of the testicular section was done in twenty high power fields (HPFs). Histological lesions were evaluated in both the tubular and extratubular compartments, using a semiquantitative method, as previously performed (Minutoli et al., 2005), to recognize lobular changes and/or interstitial extravasations. Focal or diffuse tubular changes were objectified, including germ cell detachment or loss in the tubular compartment, and coagulative necrosis. Venular and lymphatic ectasia was quantified using the ocular micrometer of the Zeiss Axioplan microscope. The greatest diameter was recorded on twenty microvessels transverse profiles for each slide as the mean ± standard deviation (SD). Histological damages were scored in accordance to the following grades: 0, absent; 1, mild; 2, moderate and 3, severe (Minutoli et al., 2005). 2.5. Immunohistochemical staining for BAX Paraffin-embedded tissues were sectioned (5 µm), and antigen retrieval was performed using microwave treatment. Tissues were treated with primary antibody against BAX (Santa Cruz Biotechnology,

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Fig. 1. Representative western blot analysis of MAPK3/MAPK1 and phosporylated MAPK3/MAPK1 (p-MAPK3/MAPK1) form in specimens of both ischemic-reperfused and contralateral testes of WT and KO mice treated with either PD98059 (5 mg/kg/i.p) or its vehicle (1 ml/kg/i.p). The upper panel shows representative autoradiography highlighting both MAPK3/MAPK1 and phosporylated MAPK3/MAPK1 (p-MAPK3/MAPK1) expression. The lower panel shows quantitative data and represents the mean ± SEM of seven experiment carried out after 30 min of reperfusion ⁎P b 0.001 vs WT testicular-I/R. §P b 0.001 vs KO testicular-I/R.

USA), diluted 1:500 and incubated overnight. Secondary antibody was provided by Dako, and the location of the reaction was visualized with 3,3'-diaminobenzidine tetra-hydrochloride (Dako cytomation Carpinteria, CA, USA) as chromogen substrate.

(1:10, v/v) solution. All substances were prepared fresh daily and administered in a volume of 1 ml/kg.

2.6. Statistical analysis

3.1. MAPK3/MAPK1 expression in testicular-I/R: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice

All data are expressed as the mean± S.E.M. Data were analyzed by ANOVA followed by post hoc evaluation. In all cases, a probability error of less than 0.05 was selected as criterion for statistical significance. For the histological results, statistical analysis was performed using a repeated measures ANOVA test with Dunn multiple comparison post test. 2.7. Drugs PD98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] was obtained from Calbiochem (San diego, California, USA). The compound was administered i.p. in dimethyl sulfoxide/0.9% NaCl

3. Results

Testicular ischemia–reperfusion injury produced a greater increase of phosphorylated MAPK3/MAPK1 (p-MAPK3/MAPK1) in KO mice than in WT animals (Fig. 1) after 30 min of reperfusion. This higher expression was evidenced in the ipsilateral testis and contralateral one (Fig. 1). No changes were observed in the total form of this kinase in both testes of either KO and WT mice (Fig. 1). Administration of PD98059, an inhibitor of MAPK3/MAPK1, immediately after the beginning of reperfusion, blunted the MAPK3/MAPK1 protein signal in the ipsilateral testis and contralateral one of both strains of animals (Fig. 1). No significant change in protein expression of MAPK3/MAPK1

Table 1 Histology of both ischemic-reperfused (I/R) and contralateral testes collected from wild type (WT) or knock out (KO) mice treated either PD98059 (5 mg/kg i.p) or its vehicle (1 ml/kg/i.p) Histopathological features

WT TI/R+ vehicle

WT TI/R KO TI/R + contralateral + vehicle vehicle

KO TI/R WT TI/R + contralateral + vehicle PD98059

WT TI/R KO TI/R + contralateral + PD98059 PD98059

KO TI/R contralateral + PD98059

Intertubular Edema Venular Ectasia Peritubular Changes Germ-cell sloughing Intraluminal Necrosis

3

1

1

1

1

0

1

1

3 3

2 1

2 0

1 0

2 1

1 0

1 0

0 0

3

0

0

0

1

0

0

0

3 (Diffuse)

1 (Focal)

0

0

0

0

0

0

Histologic grading was based on the following scale: 0 (absent), 1 (mild), 2 (moderate), 3 (severe) (N = 7).

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Fig. 2. A: Light microscopy of the ipsilateral testis collected from WT mouse undergone testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p). (H&E original magnification ×200). B: Light microscopy of the contralateral testis harvested from WT mouse subjected to testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p). (H&E original magnification ×200). C: Light microscopy of the ipsilateral testis collected from WT mouse subjected to testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). (H&E original magnification ×200). D: Light microscopy of the contralateral testis harvested WT mouse subjected to testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). (H&E original magnification ×200). E: Light microscopy of the ipsilateral testis collected from KO mouse undergone testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p). (H&E original magnification ×200). F: Light microscopy of the contralateral testis harvested KO mouse subjected to testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p). (H&E original magnification ×200). G: Light microscopy of the ipsilateral testis collected from KO mouse undergone testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). (H&E original magnification ×200). H: Light microscopy of the contralateral testis harvested KO mouse subjected to testicular ischemia– reperfusion injury and treated with PD98059 (5 mg/kg/i.p). (H&E original magnification ×200).

was detected in both testes of either Sham KO and Sham WT (results not shown).

3.3. TNF-α protein in testicular-I/R: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice

3.2. Histology: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice

WT mice had a markedly enhanced TNF-α protein in both torsioned and contralateral testes after 3 h of the reperfusion (Fig. 3). By contrast, the protein expression of this cytokine was markedly reduced in the ipsilateral and contralateral testes of KO mice after 3 h of reperfusion (Fig. 3). Administration of PD98059, an inhibitor of MAPK3/MAPK1, immediately after the beginning of reperfusion, significantly reduced the cytokine expression in the ipsilateral testis and contralateral one of both strains of mice. No significant change of TNF-α expression was detected in both testes of either Sham KO and Sham WT (results not shown).

The main histological changes observed in WT mice subjected to testicular ischemia–reperfusion injury were severe lobular haemorrhagic extravasations, venular and lymphatic vessel ectasia together with edema, germ cell hypobiosis and/or diffuse coagulative necrosis associated with high amounts of endotubular fluids (Table 1 and Fig. 2A) after 24 h of the reperfusion. Contralateral testis of WT mice subjected to ischemia–reperfusion injury showed mild microvessel ectasia. Contralateral testis did not have excess endotubular fluid or hypoxic cell changes (Table 1 and Fig. 2B) after 24 h of the reperfusion. Administration of PD98059, an inhibitor of MAPK3/MAPK1, immediately after the beginning of reperfusion, markedly attenuated the histological damage in the ipsilateral testis and contralateral one in WT animals (Table 1 and Fig. 2C and D). In agreement with our previous findings the ipsilateral testis and contralateral one of KO mice subjected to testicular ischemia– reperfusion showed a markedly blunted histological damage (Table 1 and Fig. 2E and F). Administration of PD98059, an inhibitor of MAPK3/ MAPK1, immediately after the beginning of reperfusion neither improved nor reversed the histological effects in the ipsilateral testis and contralateral testis of KO animals (Table 1 and Fig. 2G and H).

3.4. BAX expression in testicular-I/R: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice No significant difference in the basal expression of BAX was detected between testes of Sham KO and Sham WT (results not shown). Testicular ischemia–reperfusion injury produced a marked increase of BAX in both testes in WT animals (Fig. 4) following 24 h of reperfusion. By contrast BAX signal was significantly blunted in the ipsilateral and contralateral testes of KO mice (Fig. 4). Administration of PD98059, an inhibitor of MAPK3/MAPK1, immediately after the beginning of reperfusion, blunted BAX expression in the ipsilateral testis and contralateral one of both strains of animals (Fig. 4). However the effect was greater in KO mice than in WT animals.

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Fig. 3. Representative western blot analysis of TNF in specimens of both ischemic-reperfused and contralateral testes of WT and KO mice treated with either PD98059 (5 mg/kg/i.p) or its vehicle (1 ml/kg/i.p). The upper panel shows representative autoradiography highlighting TNF expression. The lower panel shows quantitative data and represents the mean ± SEM of seven experiments carried out after 180 min of reperfusion.⁎P b 0.001 vs WT testicular-I/R. §P b 0.001 vs KO testicular-I/R.

3.5. Caspase 3 and caspase 9 expression in testicular-I/R: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice No significant difference in the basal expression of caspase 3 and caspase 9 was detected between testes of Sham KO and Sham WT (results not shown). Testicular ischemia–reperfusion injury produced a marked increase in both caspase 3 and caspase 9 in both testes of WT animals (Figs.

5 and 6) following 24 h of reperfusion. By contrast caspase 3 and caspase 9 signals were significantly blunted in the ipsilateral and contralateral testes of KO mice (Figs. 5 and 6). Administration of PD98059, an inhibitor of MAPK3/MAPK1, immediately after the beginning of reperfusion, blunted the expression of caspase 3 and caspase 9 in the ipsilateral testis and the contralateral one of both strains of animals (Figs. 5 and 6). The observed effects, however, were greater in KO mice compared to WT animals.

Fig. 4. Representative western blot analysis of BAX in specimens of both ischemic-reperfused and contralateral testes of WT and KO mice treated with either PD98059 (5 mg/kg/i.p) or its vehicle (1 ml/kg/i.p). The upper panel shows representative autoradiography highlighting BAX expression. The lower panel shows quantitative data and represents the mean ± SEM of seven experiments carried out after 24 h of reperfusion. ⁎P b 0.001 vs WT testicular-I/R. §P b 0.001 vs KO TI/R. #P b 0.001 vs WT testicular-I/R +PD98059.

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Fig. 5. Representative western blot analysis of caspase 3 in specimens of both ischemic-reperfused and contralateral testes of WT and KO mice treated with either PD98059 (5 mg/kg/i.p) or its vehicle (1 ml/kg/i.p). The upper panel shows representative autoradiography highlighting BAX expression. The lower panel shows quantitative data and represents the mean ± SEM of seven experiments carried out after 24 h of reperfusion. ⁎P b 0.001 vs WT testicular-I/R. §P b 0.001 vs KO testicular-I/R. #P b 0.001 vs WT testicular-I/R +PD98059.

3.6. Immuno-histochemistry analysis for BAX in testicular-I/R: effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO and WT mice Immunohistochemical analysis revealed no significant difference in the basal protein expression of BAX between testes of Sham KO and Sham WT animals (results not shown). Testicular ischemia–reperfusion

injury, however, significantly increased immunostaining of BAX in Leydig cells for both testes of WT mice (Fig. 7A and B). KO mice subjected to testicular ischemia–reperfusion showed a weaker staining for this apoptotic protein in the Leydig cells of both testes. (Fig. 7E and F). Administration of PD98059, an inhibitor of MAPK3/ MAPK1, immediately after the beginning of reperfusion, blunted BAX

Fig. 6. Representative western blot analysis of caspase 9 in specimens of both ischemic-reperfused and contralateral testes of WT and KO mice treated with either PD98059 (5 mg/kg/i.p) or its vehicle (1 ml/kg/i.p). The upper panel shows representative autoradiography highlighting BAX expression. The lower panel shows quantitative data and represents the mean ± SEM of seven experiments carried out after 24 h of reperfusion. ⁎P b 0.001 vs WT testicular-I/R. §P b 0.001 vs KO testicular-I/R. #P b 0.001 vs WT testicular-I/R +PD98059.

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Fig. 7. A. Representative immunohistochemical BAX staining of the ipsilateral testis collected from WT mouse subjected to testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p).Immunopositivity was strongly evident in Leydig cells. (Original magnification ×200). B. Representative immunohistochemical BAX staining of the contralateral testis collected from WT mouse subjected to testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p) Immunopositivity was weak in Leydig cells. (Original magnification ×200). C. Representative immunohistochemical BAX staining of the ipsilateral testis collected from WT mouse subjected to testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). Immunopositivity was weak in Leydig cells (Original magnification ×200). D. Representative immunohistochemical BAX staining of the contralateral testis collected from WT mouse undergone testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). Immunopositivity was weak in Leydig cells (Original magnification ×200). E. Representative immunohistochemical BAX staining of the ipsilateral testis collected from KO mouse subjected to testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p).Immunopositivity was weak in Leydig cells. (Original magnification ×200). F. Representative immunohistochemical BAX staining of the contralateral testis collected from KO mouse subjected to testicular ischemia–reperfusion injury and treated with vehicle (1 ml/kg/i.p) Immunopositivity was weak in Leydig cells. (Original magnification ×200). G. Representative immunohistochemical BAX staining of the ipsilateral testis collected from KO mouse subjected to testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). Immunopositivity was weak in Leydig cells (Original magnification × 200). H. Representative immunohistochemical BAX staining of the contralateral testis collected from KO mouse undergone testicular ischemia–reperfusion injury and treated with PD98059 (5 mg/kg/i.p). Immunopositivity was weak in Leydig cells (Original magnification ×200).

immunostaining in the ipsilateral testis and the contralateral one of both strains of animals (Fig. 7C, D, G and H). 4. Discussion Torsion of the spermatic cord seriously threatens the continued viability of the ipsilateral testis. In addition function of the contralateral testis may be also damaged by unilateral torsion. The two most important factors determining testicular damage are the degree of twisting and the time period between injury and surgical repair to counter-rotate both testis and spermatic cord for inducing reperfusion. Besides resolving the acute ischemia, the surgical procedure of testicular detorsion triggers a cascade of events that may subsequently impair testicular architecture and function due to restored blood flow. The injury to the affected testis, therefore, is caused by a combination of ischemia and reperfusion that leads to production of reactive oxygen species (ROS) (Dokmeci, 2006) and to adhesion of leukocytes (Lysiak et al., 2001). Therefore, testicular torsion followed by repair produces a reperfusion injury involving complex and multifaceted molecular signalling pathways. Experimental pharmacological approaches for treatments of testicular ischemia–reperfusion injury, including the use of several

antioxidants (Wei et al., 2007; Payabvash et al., 2007; Pekcetin et al., 2007; Beheshtian et al., 2008; Nezami et al., 2008), dehydroepiandrosterone (Aksoy et al., 2007) and erythropoietin (Yazihan et al., 2007; Akcora et al., 2007) have proved to be effective. The early events which results in reperfusion injury, however, are still not fully understood. This knowledge gap weakens the potential development of new therapeutic approaches intended to be administered immediately after the surgical correction of torsional testicular injuries. Nuclear factor kappa-B (NF-κB), Mitogen-Activated Protein Kinase 3/MAPK1, MAPK8 and MAPK14 play an important role in the early stages of testicular ischaemia/reperfusion injury (TI/R) (Lysiak et al., 2003; Lysiak, 2004). We have previously found an augmented p-MAPK3/MAPK1 expression and the complete absence of p-MAPK8 and p-MAPK14 protein expression in both testes of NF-κB KO mice, when compared to WT animals. In addition, we showed a decreased TNF-α production and blunted histological damage (Minutoli et al., 2007). The reason for this modified pattern of MAPK activation in NF-κB KO animals may suggest the presence of some still unidentified mechanism that could unmask new therapeutic strategies. For example, p-MAPK3/MAPK1 complex could be over-expressed to overcome the lack of p-MAPK8, p-MAPK14 and NF-κB, with the aim

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Fig. 8. Hypothetical pathways and their putative role during testicular ischemia– reperfusion injury in mice. ↑ = increased.

of stimulating alternative inflammatory pathways. However, since KO mice have reduced TNF-α levels and reduced tissue damage, this alternative inflammatory cascade may be inefficient. Alternatively, the higher expression of p-MAPK3/MAPK1 in KO mice might suggest a protective role for this kinase. It has been shown that in myocardial ischemia–reperfusion or in the “phenomenon” of preconditioning, early activation of MAPKs may contribute to cardioprotection in the so-called reperfusion injury kinase (RISK) pathways (Hausenloy and Yellon, 2004). In order to clarify the role of MAPK3/MAPK1 up-regulation during testicular ischemia, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1 in KO mice during testicular-I/R. Administration of PD98059, abrogated MAPK3/MAPK1 expression, and slightly reduced TNF-α, but neither improved nor reversed the histological effect in KO animals. By contrast, PD98059 significantly blunted the histological damage in WT mice. This result clearly suggests that MAPK3/MAPK1 does not mediate a protective effect and therefore, rules out the hypothesis of a reperfusion injury MAPK3/MAPK1 mediated pathway operating in testicular ischemia–reperfusion injury. Indeed, the selective MAPK3/MAPK1 inhibitor succeeded in protecting the testicular damage in WT animals. This result suggests that the mitogen-activated protein kinase is likely to be involved in different inflammatory pathways that may be either NF-κB-independent or, at least in part, NF-κB-mediated depending upon the presence of the transcription factor. Reperfusion is also crucial for the activation of apoptotic events (Reed, 2000). The interaction between BCL-2 family members that promote (Bax) and suppress (Bcl-e and BcL-xL) apoptosis determines whether cells undergo programmed cell death. Furthermore, the downstream central component of the apoptotic machinery involves a family of aspartic acid-directed cysteine proteases called caspases. Caspases are expressed as inactive proenzymes and participate in a cascade triggered in response to pro-apoptotic signals (Yagmurdur et al., 2008). Among these, caspase-3 and -9 are considered to be the major executioner proteases. Apoptosis is required for normal spermatogenesis in animals and is believed to ensure cellular homeostasis and to maintain the fine balance between germ cells and Sertoli cells. Yet, apoptosis seems also to be involved in testicular ischemia–reperfusion injury. Until recently the ischemic injury of the ipsilateral twisted testis was thought to be only “necrotic” and then followed by the loss of endocrine and exocrine function. More recently, however, it has been also shown that the activation of the caspase pathway and the induction of caspase-3 and -9 proteolysis during apoptosis in the testis after ischemia (Kyriakis and Avruch, 2001). Thus, it appears that apoptosis and factors specific to programmed cell death play a central role in the pathogenesis of testicular torsion. In addition MAPKs have been shown to participate in the activation of programmed cell death.

In light of these observations, we also studied BAX, caspase-3 and -9 expression after reperfusion in both KO and WT animals. Our results suggest that testicular ischemia–reperfusion injury produced a marked increase in BAX, caspase-3 and -9 in both testes of WT animals following 24 h of reperfusion. By contrast, BAX, caspase-3 and -9 signal was significantly blunted in the ipsilateral and contralateral testes of KO mice. These results would indicate that the apoptotic machinery is activated during testicular reperfusion and that NF-κB is involved, at least in part, in programmed cell death during testicular torsion. Additionally, immunohistochemical analysis showed a greater immunostaining for BAX in the Leydig-cell in testes of WT mice subjected to testicular ischemia–reperfusion compared to KO mice suggesting that the apoptosis is acutely increased in interstitial tissue of twisted testis. Conversely, KO mice have increased MAPK3/MAPK1 expression and reduced apoptotic pathway activation. In order to understand the “significance” of MAPK3/MAPK1 overexpression in KO mice, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, during testicular-I/R. Administration of PD98059, immediately after the beginning of reperfusion, blunted BAX and caspase expression in the ipsilateral testis and contralateral one of both strains of animals. However the effects of PD98059 in KO mice was greater than in WT mice, suggesting that augmented MAPK3/MAPK1 expression may serve as an up-stream signal to turnon programmed cell death in the testis. This pathway of apoptosis may be more prominent than “necrotic-inflammatory pathway”. Therefore testicular-I/R may cause two different pathways of organ damage:1) an early pathway involving NF-κB, MAPK3/MAPK1, MAPK8, MAPK14 and TNF-α which produces an inflammatory derangement of the testis and 2) a more delayed response involving MAPK3/MAPK1 only, TNF-α, BAX, caspase-3 and -9 that likely activates apoptosis (Fig. 8). In conclusion our results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-α, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-κB independent manner. These findings should contribute to knowledge for the generation of new targeted therapies for the treatment of testicular torsion. Acknowledgements The authors are grateful to Dr. Bruce Burnett for language revision. References Akcora, B., Altug, M.E., Kontas, T., Atik, E., 2007. The protective effect of darbepoetin alfa on experimental testicular torsion and detorsion injury. Int. J. Urol 14, 846–850. Aksoy, H., Yapanoglu, T., Aksoy, Y., Ozbey, I., Turhan, H., Gursan, N., 2007. Dehydroepiandrosterone treatment attenuates reperfusion injury after testicular torsion and detorsion in rats. J. Pediatr. Surg. 42, 1740–1744. Antonuccio, P., Minutoli, L., Romeo, C., Nicotina, P.A., Bitto, A., Arena, S., Altavilla, D., Zuccarello, B., Polito, F., Squadrito, F., 2006. Lipid peroxidation activates mitogenactivated protein kinases in testicular ischemia-reperfusion injury. J. Urol. 176, 1666–1672. Barnes, P.J., 1997. Nuclear factor-kappa B. Int. J. Biochem. Cell Biol. 29, 867–870. Beheshtian, A., Salmasi, A.H., Payabvash, S., Kiumehr, S., Ghazinezami, B., Rahimpour, S., Tavangar, S.M., Dehpour, A.R., 2008. Protective effects of sildenafil administration on testicular torsion/detorsion damage in rats. World J. Urol. 26, 197–202. Dhingra, S., Sharma, A.K., Singla, D.K., Singal, P.K., 2007. p38 and ERK1/2 MAPKs mediate the interplay of TNF-alpha and IL-10 in regulating oxidative stress and cardiac myocyte apoptosis. Am. J. Physiol. Heart Circ. Physiol. 293, H3524–H3531. Dokmeci, D., 2006. Testicular torsion, oxidative stress and the role of antioxidant therapy. Folia Med. (Plovdiv) 48, 16–21. Egan, L.J., Toruner, M., 2006. NF-kappaB signaling: pros and cons of altering NF-kappaB as a therapeutic approach. Ann. N. Y. Acad. Sci. 1072, 114–122. Gaestel, M., Mengel, A., Bothe, U., Asadullah, K., 2007. Protein kinases as small molecule inhibitor targets in inflammation. Curr. Med. Chem. 14, 2214–2234. Hausenloy, D.J., Yellon, D.M., 2004. New directions for protecting the heart against ischemia–reperfusion injury: targeting the reperfusion injury salvage kinase (RISK) pathway. Cardiovasc. Res. 61, 448–460. Kyriakis, J.M., Avruch, J., 2001. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81, 807–869. Lysiak, J.J., 2004. The role of tumor necrosis factor-alpha and interleukin-1 in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis. Reprod. Biol. Endocrinol. 2, 1–10.

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